Lipopolysaccharide (LPS), derived from the outer membrane of Gram-negative bacteria, can cause a severe inflammatory response leading to sepsis and multi-organ failure. This process is particularly prevalent among surgical and trauma patients, and is a major cause of morbidity and mortality. Paradoxically, mice that harbor a signaling defect in the LPS receptor toll-like receptor 4 (TLR4) are killed by lower doses of Gram-negative bacteria than wild-type mice. This suggests that LPS can activate signaling pathways that enhance the ability of host cells to respond to pathogens. We hypothesize that LPS enhances the ability of macrophages to clear pathogens by triggering autqphagy. Autophagy is a complex lysosomal degradation pathway that can enhance the ability of macrophages to clear invading pathogens. Our preliminary studies show that LPS triggers autophagy in human monocyte derived macrophages (HMDM). Furthermore, we observed autophagosomes containing mitochondria in LPS-treated cells. Since damaged mitochondria are cleared via autophagosomes, and can trigger autophagy, we reason that LPS could trigger autophagy by altering the mitochondrial function. Published data and our own results indicate that LPS enhances macrophage resistance to several Gram- negative pathogens. It remains to be determined, however, whether the enhanced resistance is linked to autophagy. Our preliminary data and the high risk/high impact potential of this study are consistent with the criteria of the R21 funding mechanism. To pursue the aims of this proposal we have developed methodologies to interrogate the activity of HMDM at the single cell level, to express recombinant proteins in HMDM, and to suppress the expression of endogenous HMDM proteins using RNA interference. If the proposed functional link between the protective effect of LPS and autophagy is confirmed, it will impact new avenues for investigation and targets for development of pharmacological interventions that could prevent sepsis and/or improve survival. To address these issues we propose the following Specific Aims: Aim 1. To determine whether LPS-induced mitochondrial dysfunction results in HMDM autophagy. Aim 2. To determine whether autophagy enhances the resistance of LPS-treated HMDM to Gram-negative bacteria.